Detection of cellular proteins is needed for both basic life sciences research and clinical applications since proteins play essential roles in virtually each step of cellular metabolism (Wu and Singh, Curr Opin Biotechnol. 2012, 23:83-8.; Collins et al., Nature. 2003, 422:835-47; Schubert, Adv Biochem Eng Biotechnol. 2003, 83:189-209; Paez et al., Science. 2004, 304:1497-500; Soda et al., Nature. 2007; 448:561-U563; Bendall et al., Science. 2011; 332:687-696). However, proteins to be examined often have very low levels (Boschetti and Righetti, Low-Abundance Proteome Discovery: State of the Art and Protocols. 2013:1-11; Baracat-Pereira et al., Genet Mol Biol. 2012, 35:283-91; Wasinger et al., Methods Mol Biol. 2008, 424:257-75; Ackermann and Berna, Expert Rev Proteomics. 2007, 4:175-86; Ahmed and Rice, J Chromatogr B Analyt Technol Biomed Life Sci. 2005, 815:39-50). This problem may result from a small size of samples in the scenarios such as the analysis of rare circulating tumor cells, forensic specimens, and prenatal testing samples (Wang et al., Nano Today, 2013, 8:347-387; Deng et al., Sci Rep. 2014 Dec. 16; 4:7499; Zhang et al., Anal Chem. 2015, 87:9761-8; Danova et al., Expert Rev Mol Diagn. 2011, 11:473-85; Nagrath et al., Nature. 2007, 450:1235-9; Hanson and Ballantyne, Anal Biochem. 2005, 346:246-257; Denecke et al., Pediatr Res. 2005, 58:248-253; Yamamoto et al., Diagn Mol Pathol. 2004, 13:167-71). It may also result from the fact that certain proteins have a low abundance (e.g., cell membrane receptors and transcription factors) (Brewis and Brennan, Adv Protein Chem Str. 2010, 80:1-44). Their expression can be over ten orders of magnitudes lower than those highly expressed proteins (e.g., albumin in serum) (Brewis and Brennan, Adv Protein Chem Str. 2010, 80:1-44). Nevertheless their biological functions are not marginal; on the contrary, most of them make huge physiological impacts on cells at an extremely low concentration (Spitz and Furlong, Nat Rev Genet. 2012, 13:613-626). Therefore, it is important to develop highly sensitive methods for detection of proteins, particularly low-abundance proteins or those in a small sample (Wang et al., Nano letters. 2011, 11:498-504; Wang et al., ACS nano. 2011, 5:6619-6628; Crow et al., Am J Roentgenol. 2009, 192:1021-1028, Aaron et al., Nano letters. 2009, 9:3612-3618; Austin et al., J Am Chem Soc. 2011, 133(44): 17594-17597, Qian et al., J Biomed Opt. 2010, 15:046025; Seekell et al., J Biomed Opt. 2011, 16; Crow et al., ACS nano. 2011, 5:8532-8540; Kennedy et al., ACS nano. 2009, 3:2329-2339; Wang et al., Nano letters. 2012, 12:3231-3237; Fraire et al., ACS nano. 2014, 8:8942-8958).
To detect proteins, cells are often lysed to release proteins in a soluble form. Hundreds of soluble proteins can be examined quickly and precisely using mass spectrometry (MS), which has advantages such as no need to pre-label target proteins and femtomolar sensitivity in the analysis of pure proteins (Passarelli and Ewing, Curr Opin Chem Biol. 2013, 17:854-9; Jarecki et al., ACS Chem Neurosci. 2013, 4:418-34; Boggio et al., Expert Rev Proteomics. 2011, 8:591-604; Bandura et al., Anal Chem. 2009, 81:6813-22). Soluble proteins are also routinely measured with signal amplification using the enzyme-linked immunosorbent assay (ELISA) with the limit of detection at the level of pg/mL (Zhang et al., J Immunol Methods. 2011, 368:1-23; Ponde, Eur J Clin Microbiol Infect Dis. 2013, 32:985-988; Tijssen and Adam, Curr Opin Immunol. 1991, 3:233-7; Nilsson, Curr Opin Immunol. 1989, 2:898-904). ELISA can be further tuned and integrated with polymerase chain reaction (PCR) to develop immuno-PCR or nanotechnologies to develop plasmonic ELISA for ultrasensitive detection of proteins (Niemeyer et al., Nat Protoc. 2007, 2:1918-30; Sano et al., Science. 1992, 258:120-122; Burbulis et al., Nat Methods. 2005, 2:31-37; de la Rica and Stevens, Nat Nanotechnol. 2012, 7:821-824; Nam et al., Science. 2003, 301:1884-1886). Many other methods such as immunoblot analysis can also be used for detection of soluble or solubilized proteins (Hughes et al., Nat Methods. 2014, 11:749-U794; Pumford et al., Toxicol Appl Pharmacol. 1990, 104:521-32; von Wulffen et al., J Clin Pathol. 1988, 41:653-659). These highly sensitive methods require cell lysis and/or protein separation, which are not suitable for situations where whole cells are still needed during and after examination. For instance, the ability to maintain cell integrity is a prerequisite for examining location and distribution of proteins in a cell. Thus, whole-cell in situ protein analysis methods have also been rigorously studied as an alternative solution to those problems.
Immunostaining is a commonly used method for whole-cell analysis (Zola, Current protocols in cytometry/editorial board, J. Paul Robinson, 2004, Chapter 6:Unit 6 3; Turac et al., PloS one. 2013, 8; D'Hautcourt, Current protocols in cytometry/editorial board, J. Paul Robinson, 2002, Chapter 6:Unit 6 12; Fung et al., Nat Protoc. 2010, 5:357-370; Glynn and McAllister, Nat Protoc, 2006, 1:1287-1296; Perez et al., Current protocols in cytometry/editorial board, J. Paul Robinson. 2005, Chapter 6:Unit 6 20; Almeida and Bueno, Current protocols in cytometry/editorial board, J. Paul Robinson. 2001; Chapter 6:Unit 6 6). Cells are labeled with antibodies bearing fluorophores for microscopic examination or flow cytometry (Fung et al., Nat Protoc. 2010, 5:357-370; Glynn and McAllister, Nat Protoc, 2006, 1:1287-1296; Perfetto et al., Nat Rev Immunol. 2004, 4:648-55; Ullal et al., Sci Transl Med. 2014; 6; Zrazhevskiy and Gao, Nat Commun. 2013; 4:1619; Pirici et al., J Histochem Cytochem. 2009, 57: 567-575; Schweller et al., Angew Chem Int Ed Engl. 2012, 51:9292-9296). Since cell immunostaining does not need cell lysis or protein separation, sample pretreatment is relatively simple and has no problem of protein dilution. Proteins are also confined in their original locations (Huh et al., Nature. 2003, 425:686-691). If cell receptors are target proteins for examination, living cells can be directly analyzed and further used for other purposes (e.g., cell culture) afterwards. While immunostaining has been widely used for successful protein examination, spectral overlap of fluorophores is a challenging issue that often limits the measurement to a few proteins. Importantly, most of conventional methods lack the function of signal amplification.
Therefore, there is a need in the art to develop new methods that in principle have no limit of analyzing a multitude of proteins with high sensitivity. Further, there is a need for such methods to pair with protein detection methods (e.g. immunofluorescence assays) such that proteins can be detected while in their original cellular location. Such methods are particularly needed for many contemporary life science studies and clinical applications that often require comprehensive, spatially delineated analyses of complex protein pathways and molecular networks (Collins et al., Nature. 2003, 422:835-47; Zrazhevskiy and Gao, Nat Commun. 2013; 4:1619; Jaiswal et al., Nat Methods. 2004, 1:73-78; Wei et al., Angew Chem Int Ed Engl. 2014, 53:5573-5577; Howarth et al, Nat Methods. 2008, 5:397-399; Jaiswal et al, Nat Biotechnol. 2003, 21:47-51; Han et al., Nature. 2004, 430:88-93; Chen and Murphy, J Biomed Biotechnol. 2005, 2005:87-95; Taban et al., J Am Soc Mass Spectrom. 2007, 18:145-51; Altelaar et al., Nat Protoc. 2007, 2:1185-1196; McDonnell and Heeren, Mass Spectrom Rev. 2007, 26:606-43; Schubert et al., Nat Biotechnol. 2006, 24:1270-8).